The Ras-related GTP-binding protein Cdc42 has been implicated in a number of fundamentally important cellular processes including the establishment of cell polarity and motility through influences on the actin cytoskeleton, and the regulation of cell-cycle progression. The tight regulation of the GTP-binding/GTPase cycle of Cdc42 is essential to its cellular functions. Mutations that trap Cdc42 in either the GDP-bound or GTP-bound state inhibit cell growth, whereas those that give rise to an accelerated cycling of Cdc42 between these states lead to cellular transformation and tumor formation in nude mice. During the past funding period, we have combined biochemical, genetic, and structural biology-based approaches to study the regulation of Cdc42 by guanine nucleotide exchange factors and the Rho-GDP-dissociation inhibitor (RhoGDI), as well as examined the interactions of activated Cdc42 with various downstream signaling targets, including ACK (Activated Cdc42- associated kinase) and IQGAP. In addition, we have identified two new targets for Cdc42, the gamma-coatomer subunit (gammaCOP) of the COPI complex and the p85Cool-1 (for Cloned-out of library)/beta-Pix (PAK-interactive exchange factor) protein. These studies have provided us with new information regarding the molecular mechanisms underlying the cellular regulation and function of Cdc42. However, perhaps most important, work during the past funding period has raised an interesting and somewhat unanticipated role for Cdc42, namely linking intracellular trafficking activities to cell signaling and cell growth regulation. In this renewal application, we plan to examine this interesting new role for Cdc42 by focusing on those upstream regulators and downstream target/effectors that appear to implicate most strongly Cdc42 in cellular trafficking functions. This will constitute 4 lines of study. Aim la.) Identify and characterize the protein complexes that couple the activation of Cdc42 to the sorting and trafficking of EGF receptors. Aim lb.) Determine why Cdc42-gammaCOP interactions are essential for Cdc42-mediated cellular transformation. Aim 2a.) Determine whether IQGAP serves as a docking site for Cdc42 and other proteins involved in intracellular trafficking functions. Aim 2b.) Determine whether RhoGDI plays a fundamental role in Cdc42-mediated intracellular trafficking activities. These studies are expected to yield important new insights into a critical cellular function for Cdc42 that may form the basis for a diversity of Cdc42-mediated cellular responses ranging from cell cycle progression and cellular transformation to morphological and actin cytoskeletal changes.